Cervical cancer is the second most common cause of cancer deaths in women worldwide, with about a half million new cases and about a quarter of a million deaths every year. In the US, the cervical cancer mortality rate has decreased substantially due to the success of cervical cancer screening programs, which detect precancers and lead to intervention and treatment of precancers before they develop into cancer. The current paradigm for cervical cancer screening is based on the Pap test, which is a cytologically based test of cells scraped from the cervix and examined microscopically either by a human or by a machine to detect changes indicating dysplastic cell growth. The test is subjective with significant inter-observer variability, and is limited by low sensitivity and high false positive results. Reports of false-negative rates in cervical cytology have varied widely, from as low as 1.6% to almost 28%. About 4 million abnormal Pap tests are diagnosed in the United States each year as atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL), or atypical glandular cells (AGC).
Under current practice guideline, these cases are referred for colposcopy to further identify the subset of patients that will have clinically significant high-grade lesions (CIN2/3) or endocervical neoplasia on cervical biopsy. It was reported that patients with a cytologic diagnosis of ASC-US (over 2 million cases annually in the US) have only 5% to 17% chance of underlying CIN2/3 on cervical biopsy, and in LSIL (about 1.6 million cases in the US annually), CIN2/3 was found in up to 25%. These data suggest that about 3 million of cases with ASC-US or LSIL on Pap, colposcopy is unnecessary. Although colposcopic biopsy has historically been considered the gold standard, recent reports indicate that cervical biopsies may miss 33% to 50% of high-grade disease because of sampling or diagnostic errors. As a result, it may be difficult to differentiate between false positive cervical cytology results versus false-negative biopsy results. Therefore, there is strong need for a test to identify high-grade dysplasia to triage the patient who can benefit most from intervention.
Although most low grade cervical dysplasias spontaneously regress without ever leading cervical cancer, dysplasia can serve as an indication that increased vigilance is needed. CIN1 is the most common and most benign form of cervical intraepithelial neoplasia and usually resolves spontaneously within two years. Because of this, LSIL results can be managed with a simple “watch and wait” philosophy. However, because there is a 12-16% chance of progression to more severe dysplasia, the physician may want to follow the results more aggressively by performing a colposcopy with biopsy. If the dysplasia progresses, treatment may be necessary. Therefore, what is needed is a method to detect HPV oncoproteins in situ. It would be particularly helpful in ASC-US or LSIL, or CIN1 patients to detect high-grade dysplasia cells and to identify those underlying CIN2 or above who may benefit immediate intervention, and avoid anxiety for “wait and see”.
Infection by Human Papillomaviruses (HPV) at specific epithelium cells to induce epithelial proliferations plays an important role for cervical carcinogenesis. About 99 percent of confirmed cervical cancer cases are found to be associated with HPV infection with biopsy-confirmed squamous intraepithelial lesions (SIL) or cervical intraepithelial neoplasia (CIN). The incidence of HPV infection, primarily transmitted through sexual contact, is highest among young women and about 20 millions of sexually active men and women worldwide are currently infected. Approximately 1% of the population has genital warts and 4% of women have cervical precancerous lesions, such as low grade of squamous intraepithelial lesion (LSIL) or high grade of squamous intraepithelial lesion (HSIL) or atypical squamous cells of undetermined significance (ASC-US).
The presence of these lesions, preferentially observed in women aged 35-40 yrs, are at high risk of progression toward invasive cervical cancer. It is general thought that persistent infection of human Papillomavirus (HPV) is essential for developing precancerous epithelial lesions. Infection of high-risk types HPV for women with LSIL may or may not progress to HSIL. In fact, remission occurs in majority of LSIL human subjects while some progress to HSIL. Although 99.7% of cervical cancers are HPV positive, integration of viral genome into the host genome is required to facilitate the necessary genes to express for developing into HSIL or cancer. In fact, only one in every 10 women with persistent HPV infection may develop into higher grades of CIN lesions, such as cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2, and CIN3, respectively), and a portion of these epithelial lesion cases may ultimately progress into cervical cancer.
Disease stages caused by HPV infection include an early stage HPV infection, a late stage HPV infection, Atypical squamous cells of undetermined significance (ASC-US), Atypical squamous cells, cannot exclude HSIL (ASC-H), Atypical glandular cells (AGC), low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), cervical intraneoplasm CIN1, CIN2, CIN3 representing a mild, moderate, or severe cell dysplasia respectively, invasive cervical cancer, adenocarcinoma, or squamous cell carcinoma (SCC).
In the past, screening for cervical cancer is based on conventional cytology by Papanicolaou (Pap) smear and suspicious smears are followed up with colposcopy, and/or histological biopsy. The use of cytological screening leads to a remarkable reduction in the mortality of cervical cancer. However, due to subjective test criteria, drawbacks of Pap smear tests include difficulty in obtaining samples, poor inter- and intra-observer agreement, a high rate of false negatives (up to 20%) and false positive, the requirements for specialized laboratories staffed with highly trained personnel, and inability to identify a large proportion of HPV-infected persons. More reproducible assays are needed to improve the current screening method to avoid unnecessary medical intervention and psychological distress for the affected women. The current cervical cytology screening has sensitivity ranged from 30% to 87% and specificity ranged from 86% to 100%.
Detecting HPV infection by nucleic acid methods, has been developed, but not ideal, due to not only its high cost, assay operation procedures, the requirements for facility, equipment, and highly trained personnel, but also its very low positive predictive value to CIN. In addition, DNA testing could not differentiate the diagnosis of LSIL from HSIL, nor CIN lesions from non-transforming latent or remissive viral infection. Assay for the detection of E6/E7 mRNA suggested equivalent sensitivity to HPV DNA testing with higher positive predictive value. However, there are limited reports showing direct detection of E6/E7 oncoproteins in situ. What is needed is a low cost, simple, sensitive and specific assay that can be performed on routine practice of a clinical lab or doctor office and capable of detecting early stage of epithelial lesions, distinguish LSIL from HSIL, or predicting the risk of progression into cervical cancer.
Known protocols for the production of monoclonal antibodies to HPV are generally unsuitable for the production of anti-HPV monoclonal antibodies and cannot be used in immunocytochemical diagnostic tests for screening general human population. This is because antibodies produced by these protocols will not necessarily react with the naturally occurring HPV protein in infected human cells. In addition, the epitopes recognized by prior antibodies will not necessarily be those epitopes which are resistant to the standard procedures involved in the sampling, fixing and storing of clinical specimens. Other attempts to detect the presence of HPV related antibodies or viral proteins in a human subject by ELISA (enzyme linked immunosorbent assays) generally lead to extremely low assay sensitivity and thus can not be developed into a commercially suitable diagnostic test. Most of these ELISA assays target a single viral protein or short peptide fragments, which are not able to interact well or bind strongly and specifically to antibodies from the human subject. The assay specificity and sensitivity are so low that even using samples from patients confirmed with HPV associated invasive cervical cancer, only 53% of the patient samples were found positive for HPV infection. Given the testing populations come from general screening, with or without low grade, or precancerous, the sensitivity of the assay will be too low to apply for clinical practice. Thus, there is no successful ELISA assay available as a diagnostic tool for clinical samples.
There are only about 15 types out of more than 100 types of HPV infection considered to become high-risk of developing into CIN or cervical cancer. Also, around 70% of cervical cancer cases and 50% of CIN2 and CIN 3 cases are attributed to high risk HPV type-16 and HPV type-18 infections. However, some progressive cervical cancer cases are related to infection by low risk HPV types, while infection of some HPV types will never progress into cervical cancer. It becomes important to identify those HPV infections with particular oncogenic proteins expression rather than just identify high risk type(s) of HPV infection. Thus, there is a need for detecting HPV oncoproteins as cervical cancer biomarkers to better identify the risk for developing HSIL, or precancerous, or cervical cancer.
Developing appropriate assays, such as HPV immunoassays, is needed for detection of such HPV oncoproteins or biomarkers for cervical cancer. The presence of E6/E7 oncoproteins in CIN 2 and CIN3 lesions could be evidence to indicate high risk of progression. However, there is limited antibody available for the detection of E6/E7 oncoprotein in situ. Therefore, there is a need to develop antibodies and immunological assays for detecting HPV oncoproteins as cervical cancer biomarkers to identify HSIL or ≧CIN2 (CIN2 and above), or precancerous to screen for invasive cervical cancer and/or the risk for malignant transformation into cervical cancer and other HPV associated cancers.